[mira_talk] Re: tweaking Manifest for polyploid genome
- From: "Gutierrez, Juan" <Juan.Gutierrez@xxxxxxxxxxxx>
- To: "mira_talk@xxxxxxxxxxxxx" <mira_talk@xxxxxxxxxxxxx>
- Date: Mon, 24 Feb 2014 22:37:23 +0000
Thanks Adrian,
The illumina coverage is 80-100x, so pretty decent. I did an assembly with only
illumina reads and yes the numbers were better but not fully recovering the
genes and shuffling snps among the 3 homologous genes. Thus, I thought that
adding long 454 reads could help in resolving gene copies to their full extent.
Paradoxically, I am also getting better assemblies using 454 reads alone, it is
just that I can't get both types of reads assembled together.
Juan
-----Original Message-----
From: mira_talk-bounce@xxxxxxxxxxxxx [mailto:mira_talk-bounce@xxxxxxxxxxxxx] On
Behalf Of Adrian Pelin
Sent: Monday, February 24, 2014 11:49 AM
To: mira_talk@xxxxxxxxxxxxx
Subject: [mira_talk] Re: tweaking Manifest for polyploid genome
What is your coverage for the illumina PE reads? In my experience, 454 +
illumina assemblies with MIRA are worse in stats than illumina assemblies
alone. If your illumina coverage is enough, try using it only without 454.
On 2/24/2014 11:30 AM, Gutierrez, Juan wrote:
> Thanks Hanquan and Martin for your answers.
>
> There are 3 types of reads in the assembly:
>
> 869,000 454 reads, about 500bp long on average.
> 32,254,100 illumina 100bp pair ended reads (approx total 62 million
> reads)
> 3,897,411 illumina 100bp single reads (result of the trimming process
> where the other pair was of bad quality)
>
> All reads are longer than 90bp (I removed the ones shorter than that
> after trimming)
>
> I am pretty confident on the trimming I did and all remaining reads are of
> extreme high quality. I agree with Martin in that reads do not map, but I
> think it is because of the similarity among the 3 different copies of each
> gene. They just do not know where to map unequivocally.
>
> I am thinking that I might have been very strict on the parameters. Maybe the
> right approach for this kind of repetitive assembly is being less strict on
> the parameters and let Mira resolve the uncertainties?
>
> Juan
>
>
> -----Original Message-----
> From: mira_talk-bounce@xxxxxxxxxxxxx
> [mailto:mira_talk-bounce@xxxxxxxxxxxxx] On Behalf Of Martin Mokrejs
> Sent: Monday, February 24, 2014 8:16 AM
> To: mira_talk@xxxxxxxxxxxxx
> Subject: [mira_talk] Re: tweaking Manifest for polyploid genome
>
> Hi Juan,
> how many 454 reads do you have on input? I see max coverage 180830 for the
> 454 technology. also from "Coverage assessment" I see that only 454 is
> covered at 0.78x and Solexa at 28.34x. Let me suspect your main issue is
> proper trimming of raw reads. Looks the reads just do not assemble or you
> sequenced too few material using 454. Ah, I see in your Manifest file you
> used RapidLib approach and MIDs ... poor boy, how did you remove them?
>
> Maybe you would appreciate as a paid service my help, please see
> http://www.bioinformatics.cz . There are plenty of tricks needed to
> get
> 454 trimming right and I don't know any other tool (except mine) ;) doing
> that right, not even of a tool doing the proper queries for all adapters,
> primers, artifacts. However, a lot of effort had to be put into the wrapper
> code to manage and interpret the candidate alignments.
> Having just the right queries is not enough. 3 years of work, 25k lines of
> code in python. My apologies if this this is considered as an Ad, I couldn't
> resist.
> Martin
>
>
> Gutierrez, Juan wrote:
>> Hi,
>>
>>
>>
>> I am trying to do RNA-seq de novo on a polyploid (hexaploid) genome
>> using a combination of 454 and illumina 100bp paired ended reads. The
>> three copies of each gene are highly similar to one another. I am
>> having trouble in separating each one of the three copies into three
>> different fully-length assemblies. Most of the times I just get a
>> fragment of each of the three copies. I am guessing that when Mira
>> finds a difference between highly similar transcripts, it just can’t
>> assess if there is a polymorphism between 2 of the copies or a
>> sequencing error. In any case, Mira seems to end the assembly way
>> before reaching the end of the transcript.
>>
>>
>>
>> I have prepared and run 2 different Manifests. I am getting better
>> results with Manifest.conf (less number of contigs but longer) than
>> with Manifest2.conf (higher number but shorter contigs), so I am
>> supposing that I could eventually separate the 3 copies of each gene
>> by fine-adjusting the parameters.
>>
>>
>>
>> Any suggestion would be greatly appreciated,
>>
>> Thanks so much!
>>
>> Juan
>>
>>
>>
>>
>>
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